Method for purifying rna

ABSTRACT

The present invention relates to methods for purifying RNA by chromatography under high salt conditions, e.g. by hydrophobic interaction chromatography.

This application is a continuation of U.S. application Ser. No.16/464,152, filed May 24, 2019, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/EP2017/080703, filed Nov. 28, 2017, which claims benefit ofInternational Application No. PCT/EP2016/079026, filed Nov. 28, 2016,the entire contents of each of which are hereby incorporated byreference.

This invention was made with government support under HR0011-11-3-0001awarded by the Defense Advanced Research Projects Agency. The governmenthas certain rights in the invention.

The instant application contains a Sequence Listing, which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 9, 2022, isnamed CRVCP0236USC1_ST25.txt and is 1.2 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates to methods for purifying RNA bychromatography under high salt conditions, e.g. by hydrophobicinteraction chromatography.

BACKGROUND OF THE INVENTION

RNA is emerging as an innovative candidate for a variety ofpharmaceutical applications, but efficient purification of RNA is stilla challenge. This is partly due to the different types and combinationsof undesired contaminants in a sample that need to be separated from adesired RNA species to obtain a pure RNA sample. Such contaminants aretypically components and by-products of any upstream processes, forexample RNA manufacture. If RNA in vitro transcription is used toproduce large RNA molecules, the sample after transcription typicallycontains the desired RNA species and various contaminants such asundesired RNA species, various proteins, spermidine, DNA template orfragments thereof, pyrophosphates, free nucleotides, endotoxins,detergents, and organic solvents.

Commercial downstream applications (e.g. formulation procedures and/oruse as a pharmaceutical composition and/or vaccine) pose furtherconstraints on any purification method for RNA requiring (i) a highdegree of purity while retaining RNA stability and functionality; (ii)compatibility with any formulation requirements of the RNA for in vivodelivery; and (iii) compliance with good manufacturing practices.Furthermore, in order to meet industrial applicability, any RNApurification method must enable consistent, cost- and time-efficient, aswell as quick, easy, reproducible, repetitive, cleanable (cleaning-inplace), and scalable (large scale, small scale) operation.

A common laboratory technique is RNA precipitation, allowing for sampleconcentration as well as depletion of contaminating high molecularweight contaminants and low molecular weight contaminants such asproteins and spermidine, respectively. However, precipitation is not themethod of choice in industrial production processes since precipitationand re-solubilisation of nucleic acids is time-consuming. Moreover, theuse of alcohols and other organic solvents should be avoided in a highlyregulated environment, e.g. current good manufacturing processes (cGMP).

Moreover, the use of silica-based columns for RNA purification has thedisadvantage that silica based materials do not allow cleaning withcommon cleaning solutions such as NaOH etc. as silica materials are notcompatible with alkaline buffers commonly used for cleaning (cleaning inplace).

Other processes for the purification of RNA are described in the art asoutlined below.

WO 03/051483 A1 describes a method for purifying a polynucleotide by achromatographic process comprising a combination of steps which arebased on different chromatographic principles, such as hydrophobicinteraction chromatography, polar interaction chromatography and anionexchange chromatography.

WO 2008/077592 discloses a method for purifying RNA on a preparativescale with ion-pairing reverse phase HPLC using a porous reversedstationary phase. It is reported that a particular advantage of usingthe specified porous stationary phase is that excessively high pressurescan be avoided, facilitating a preparative purification of RNA.

WO 2014/140211, WO 2014/152966 and PCT/EP2016/062152 disclose methods ofpurifying RNA by means of tangential flow filtration. However, such amethod is only suitable for large-scale preparations and technically notappropriate for small scale-preparations.

Hence, there remains a need for further RNA purification methods, and inparticular, for those that allow cost- and time-efficient purificationof RNAs at various scale with high yield and pharmaceutical-gradepurity, stability, and shelf life. Said further purification methodsshould ideally allow for a cleaning of the RNA preparation (e.g.,depletion of contaminants from crude preparations), for a polishing ofRNA preparations (e.g., depletion of residual contaminants such assolvents etc. from purified RNA preparation), for a concentration of theRNA preparation, for capturing an RNA of an RNA preparation, and for aconditioning of the RNA preparation (e.g., re-buffering). In particular,methods are required that are executable in a regulated environment(e.g., cGMP) and that are scalable, allowing for both small-scale andlarge-scale RNA preparations. Specifically, methods are needed to allowRNA purification in a small-scale manufacturing process that can be e.g.used in high throughput screening approaches or in the production ofsmall amounts of pharmaceutical-grade RNA e.g. for personalizedtherapies. Further, methods are needed to allow RNA purification usingmaterials compatible with common alkaline cleaning solutions. Forlarge-scale preparations the method should allow for operations at largeflow rates.

It is thus an object of the present invention to provide further RNApurification methods.

SUMMARY OF THE INVENTION

The inventors surprisingly found that applying a crude RNA in vitrotranscription reaction mixture (including enzymes and proteins such asRNA polymerase, spermidine, desired RNA products, abortive RNA products,DNA template, NTPs etc.) or HPLC purified RNA under high salt conditionsto a monolithic column with hydroxyl ligands led to binding of thedesired RNA to the respective column support material and to depletionof undesired contaminants (enzymes, proteins etc.).

In addition, the inventors surprisingly found that applying HPLCpurified RNA under high salt conditions to a column having a sulfate(SO₃) ligand which column is typically used for cation exchangechromatography also led to binding of the desired RNA to the respectivecolumn support material and to depletion of undesired contaminants(spermidine etc.).

Hence, the method of the present invention may be used for purifyingand/or re-buffering and/or concentrating and/or polishing and/orcapturing of a crude in vitro transcription mixture, an eluate from aRP-HPLC column containing RNA, or already purified RNA (e.g., HPLCpurified RNA) or other RNA preparations (e.g., cellular RNApreparations).

Accordingly, the present invention relates to a method for purifyingRNA, comprising the steps of:

a) applying a sample containing RNA in an equilibration buffer having ahigh salt concentration to a support material capable of binding the RNAunder high salt conditions, wherein the support comprises hydroxyl orsulfate groups;

b) optionally, washing the support material with a washing buffer havinga high salt concentration; and

c) eluting the nucleic acid from the support material with an elutionsolution.

In one embodiment, the method does not comprise a polar interactionchromatography or an anion exchange chromatography step.

Preferably, the RNA is in vitro transcribed RNA.

In one embodiment, the equilibration buffer and/or the washing bufferhas a salt concentration of 50 mM to 5M.

In one embodiment, the equilibration buffer and/or the washing buffercomprises sodium chloride or ammonium sulfate.

In one embodiment, the equilibration buffer and/or the washing buffercomprises 2 M NaCl.

In one embodiment, the equilibration buffer and/or the washing buffercomprises 20 mM HEPES-NaOH, pH 7.0, 2 M NaCl.

In one embodiment, the equilibration buffer and the washing buffer havethe same composition and the same pH.

The support material may be a monolithic support material.

In one embodiment the support material is a methacrylate polymer.

The hydroxyl ligand or sulfate moiety may be attached directly to thesupport material.

The RNA may be eluted by gradually decreasing the salt concentration.

In one embodiment the elution solution does not contain a salt.

In one embodiment the elution solution comprises 20 mM HEPES-NaOH, pH7.0.

The present invention further relates to a method for purifying in vitrotranscribed RNA, comprising the steps of:

a) transcribing RNA from a template DNA in vitro;

b) applying a sample containing the in vitro transcribed RNA in anequilibration buffer having a high salt concentration to a supportmaterial capable of binding the RNA under high salt conditions;

c) optionally, washing the support material with a washing buffer havinga high salt concentration; and

d) eluting the RNA from the support material with an elution solution.

The method may further comprise a step al) of degrading the templateDNA, wherein the template DNA may be degraded by treatment with DNase.

The method may further comprise a step a2) of subjecting the in vitrotranscribed RNA to an RP-HPLC step and/or a step e) of preparing apharmaceutical composition comprising said RNA.

In one embodiment the equilibration buffer and/or the washing buffer hasa salt concentration of 50 mM to 5M.

In one embodiment the equilibration buffer and/or the washing buffercomprises sodium chloride or ammonium sulfate.

In one embodiment the equilibration buffer and/or the washing buffercomprises 2 M NaCl.

In one embodiment the equilibration buffer and/or the washing buffercomprises 20 mM HEPES-NaOH, pH 7.0, 2 M NaCl.

In one embodiment the equilibration buffer and the washing buffer havethe same composition and the same pH.

The support material may be a monolithic support material.

The support material may be a methacrylate polymer.

Preferably, the support material comprises a ligand capable of bindingthe RNA and the ligand may be a hydroxyl ligand or a sulfate moiety.Preferably, the hydroxyl ligand or sulfate moiety is attached directlyto the support material.

The RNA may be eluted by gradually decreasing the salt concentration.

In one embodiment the elution solution does not contain a salt.

In one embodiment the elution buffer comprises 20 mM HEPES-NaOH, pH 7.0.

The present invention also relates to a method for purifying in vitrotranscribed RNA, comprising the steps of:

a) transcribing RNA from a template DNA in vitro;

b) degrading the template DNA;

c) subjecting the in vitro transcribed RNA to an RP-HPLC step;

d) applying the eluate from the RP-HPLC in an equilibration bufferhaving a high salt concentration to a support material capable ofbinding the RNA under high salt conditions;

e) washing the support material with a washing buffer having a high saltconcentration; and

f) eluting the RNA from the support material with an elution solution.

The method may further comprise a step g) of preparing a pharmaceuticalcomposition comprising said RNA.

The template DNA may be degraded by treatment with DNase.

In one embodiment the equilibration buffer and/or the washing buffer hasa salt concentration of 50 mM to 5M.

In one embodiment the equilibration buffer and/or the washing buffercomprises sodium chloride or ammonium sulfate.

In one embodiment the equilibration buffer and/or the washing buffercomprises 2 M NaCl.

In one embodiment the equilibration buffer and/or the washing buffercomprises 20 mM HEPES-NaOH, pH 7.0, 2 M NaCl.

In one embodiment the equilibration buffer and the washing buffer havethe same composition and the same pH.

The support material may be a monolithic support material.

The support material may be a methacrylate polymer.

Preferably, the support material comprises a ligand capable of bindingthe RNA and the ligand may be a hydroxy ligand or a sulfate moiety.Preferably, the hydroxyl ligand or sulfate moiety is attached directlyto the support material.

The RNA may be eluted by gradually decreasing the salt concentration.

In one embodiment the elution solution does not contain a salt.

In one embodiment the elution buffer comprises 20 mM HEPES-NaOH, pH 7.0.

The present invention also relates to a method for purifying in vitrotranscribed RNA, comprising the steps of:

a) transcribing RNA from a template DNA in vitro;

b) degrading the template DNA;

c) subjecting the in vitro transcribed RNA to an RP-HPLC step;

d) removing organic solvent from the eluate of the RP-HPLC step;

e) applying the purified RNA in an equilibration buffer having a highsalt concentration to a support material capable of binding the RNAunder high salt conditions;

f) washing the support material with a washing buffer having a high saltconcentration; and

g) eluting the RNA from the support material with an elution solution.

The method may further comprise a step h) of preparing a pharmaceuticalcomposition comprising said RNA.

The template DNA may be degraded by treatment with DNase.

In one embodiment the equilibration buffer and/or the washing buffer hasa salt concentration of 50 mM to 5M.

In one embodiment the equilibration buffer and/or the washing buffercomprises sodium chloride or ammonium sulfate.

In one embodiment the equilibration buffer and/or the washing buffercomprises 2 M NaCl.

In one embodiment the equilibration buffer and/or the washing buffercomprises 20 mM HEPES-NaOH, pH 7.0, 2 M NaCl.

In one embodiment the equilibration buffer and the washing buffer havethe same composition and the same pH.

The support material may be a monolithic support material.

The support material may be a methacrylate polymer.

Preferably, the support material comprises a ligand capable of bindingthe RNA and the ligand may be a hydroxy ligand or a sulfate moiety.Preferably, the hydroxyl ligand or sulfate moiety is attached directlyto the support material.

The RNA may be eluted by gradually decreasing the salt concentration.

In one embodiment the elution solution does not contain a salt.

In one embodiment the elution buffer comprises 20 mM HEPES-NaOH, pH 7.0.

The present invention further relates to a method for purifying in vitrotranscribed RNA, comprising the steps of:

a) transcribing RNA from a template DNA in vitro;

b) applying a sample containing the in vitro transcribed RNA in anequilibration buffer comprising 20 mM HEPES-NaOH, pH 7.0 and 2 M NaCl toa monolithic support comprising a hydroxyl or a sulfate moiety;

c) washing the support material with said equilibration buffer; and

d) eluting the RNA from the support material by a gradually decreasingsalt gradient using an elution buffer comprising 20 mM HEPES-NaOH, pH7.0.

The present invention also relates to a method for purifying in vitrotranscribed RNA, comprising the steps of:

a) transcribing RNA from a template DNA in vitro;

b) degrading the template DNA by DNase treatment;

c) subjecting the in vitro transcribed RNA to an RP-HPLC step;

d) applying the eluate from the RP-HPLC in an equilibration buffercomprising 20 mM HEPES-NaOH, pH 7.0 and 2 M NaCl to a monolithic supportcomprising a hydroxyl or a sulfate moiety;

e) washing the support material with said equilibration buffer; and

f) eluting the RNA from the support material by a gradually decreasingsalt gradient using an elution buffer comprising 20 mM HEPES-NaOH, pH7.0.

The method may further comprise a step g) of preparing a pharmaceuticalcomposition comprising said RNA.

The present invention also relates to a method for purifying in vitrotranscribed RNA, comprising the steps of:

a) transcribing RNA from a template DNA in vitro;

b) degrading the template DNA by DNase treatment;

c) subjecting the in vitro transcribed RNA to an RP-HPLC step;

d) removing organic solvent from the eluate of the RP-HPLC step;

e) applying the eluate from the RP-HPLC in an equilibration buffercomprising 20 mM HEPES-NaOH, pH 7.0 and 2 M NaCl to a monolithic supportcomprising a hydroxyl or a sulfate moiety;

f) washing the support material with said equilibration buffer; and

g) eluting the RNA from the support material by a gradually decreasingsalt gradient using an elution buffer comprising 20 mM HEPES-NaOH, pH7.0.

The method may further comprise a step h) of preparing a pharmaceuticalcomposition comprising said RNA.

Definitions

For the sake of clarity and readability, the following definitions areprovided. Any technical feature mentioned for these definitions may beread on each and every embodiment of the invention. Additionaldefinitions and explanations may be specifically provided in the contextof these embodiments. Unless defined otherwise, all technical andscientific terms used herein generally have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Generally, the nomenclature used herein and the laboratoryprocedures in cell culture, molecular genetics, organic chemistry, andnucleic acid chemistry and hybridization are those well-known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. The techniques and procedures are generallyperformed according to conventional methods in the art and variousgeneral references (e.g., Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, 2d ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.), which are provided throughout this document.

Purification: The term “purification” or “purifying” is understood tomean that the desired RNA in a sample is separated and/or isolated fromthe impurities present therein. Thus, after subjecting the RNA to themethod of the present invention the RNA is present in a purer form thanin the RNA-containing sample before subjecting it to the method of thepresent invention. Undesired constituents of RNA-containing sampleswhich therefore need to be separated may in particular be enzymes suchas RNA polymerase, other proteins, spermidine, and nucleotides.

Using the method according to the invention, RNA is purified which has ahigher purity after purification than the starting material. It isdesirable in this respect for the degree of purity to be as close aspossible to 100%. A degree of purity of more than 70%, in particular80%, very particularly 90% and most favorably 99% or more may beachieved in this way. The degree of purity may for example be determinedby an analytical HPLC, wherein the percentage provided above correspondsto the ratio between the area of the peak for the target RNA and thetotal area of all peaks representing the by-products.

RNA, mRNA: RNA is the usual abbreviation for ribonucleic acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotide monomers.These nucleotides are usually adenosine-monophosphate (AMP),uridine-monophosphate (UMP), guanosine-monophosphate (GMP) andcytidine-monophosphate (CMP) monomers or analogs thereof, which areconnected to each other along a so-called backbone. The backbone isformed by phosphodiester bonds between the sugar, i.e. ribose, of afirst and a phosphate moiety of a second, adjacent monomer. The specificorder of the monomers, i.e. the order of the bases linked to thesugar/phosphate-backbone, is called the RNA sequence. Usually RNA may beobtainable by transcription of a DNA sequence, e.g., inside a cell. Ineukaryotic cells, transcription is typically performed inside thenucleus or the mitochondria. In vivo, transcription of DNA usuallyresults in the so-called premature RNA which has to be processed intoso-called messenger-RNA, usually abbreviated as mRNA. Processing of thepremature RNA, e.g. in eukaryotic organisms, comprises a variety ofdifferent posttranscriptional-modifications such as splicing,5′-capping, polyadenylation, export from the nucleus or the mitochondriaand the like. The sum of these processes is also called maturation ofRNA. The mature messenger RNA usually provides the nucleotide sequencethat may be translated into an amino acid sequence of a particularpeptide or protein. Typically, a mature mRNA comprises a 5′-cap,optionally a 5′UTR, an open reading frame, optionally a 3′UTR and apoly(A) sequence.

In addition to messenger RNA, several non-coding types of RNA existwhich may be involved in regulation of transcription and/or translation,and immunostimulation. The term “RNA” further encompasses RNA molecules,such as viral RNA, retroviral RNA and replicon RNA, small interferingRNA (siRNA), antisense RNA, CRISPR/Cas9 guide RNA, ribozymes, aptamers,riboswitches, immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA(rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA),microRNA (miRNA), and Piwi-interacting RNA (piRNA). Further the term mayencompass circular RNA (circRNA), wherein the circRNA is preferably aprotein-coding circRNA.

Modified nucleoside triphosphate: The term “modified nucleosidetriphosphate” as used herein refers to chemical modifications comprisingbackbone modifications as well as sugar modifications or basemodifications. These modified nucleoside triphosphates are herein alsocalled (nucleotide) analogs.

In this context, the modified nucleoside triphosphates as defined hereinare nucleotide analogs/modifications, e.g. backbone modifications, sugarmodifications or base modifications. A backbone modification inconnection with the present invention is a modification, in whichphosphates of the backbone of the nucleotides are chemically modified. Asugar modification in connection with the present invention is achemical modification of the sugar of the nucleotides. Furthermore, abase modification in connection with the present invention is a chemicalmodification of the base moiety of the nucleotides. In this contextnucleotide analogs or modifications are preferably selected fromnucleotide analogs which are applicable for transcription and/ortranslation.

Sugar Modifications

The modified nucleosides and nucleotides, which may be used in thecontext of the present invention, can be modified in the sugar moiety.For example, the 2′ hydroxyl group (OH) can be modified or replaced witha number of different “oxy” or “deoxy” substituents. Examples of “oxy”-2′ hydroxyl group modifications include, but are not limited to, alkoxyor aryloxy (—OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroarylor sugar); polyethyleneglycols (PEG), —O(CH₂CH₂O)nCH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; and aminogroups (—O-amino, wherein the amino group, e.g., NRR, can be alkylamino,dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, ordiheteroaryl amino, ethylene diamine, polyamino) or aminoalkoxy.

“Deoxy” modifications include hydrogen, amino (e.g. NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, or amino acid); or the amino group can be attachedto the sugar through a linker, wherein the linker comprises one or moreof the atoms C, N, and O.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified nucleotide can include nucleotidescontaining, for instance, arabinose as the sugar.

Backbone Modifications

The phosphate backbone may further be modified in the modifiednucleosides and nucleotides. The phosphate groups of the backbone can bemodified by replacing one or more of the oxygen atoms with a differentsubstituent. Further, the modified nucleosides and nucleotides caninclude the full replacement of an unmodified phosphate moiety with amodified phosphate as described herein. Examples of modified phosphategroups include, but are not limited to, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. The phosphate linker can also be modified by thereplacement of a linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates).

Base Modifications

The modified nucleosides and nucleotides, which may be used in thepresent invention, can further be modified in the nucleobase moiety.Examples of nucleobases found in RNA include, but are not limited to,adenine, guanine, cytosine and uracil. For example, the nucleosides andnucleotides described herein can be chemically modified on the majorgroove face. In some embodiments, the major groove chemicalmodifications can include an amino group, a thiol group, an alkyl group,or a halo group.

In some embodiments, the nucleotide analogs/modifications are selectedfrom base modifications, which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-Iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,l-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-l-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine,2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface and can include replacing hydrogen on C-5 of uracil with a methylgroup or a halo group.

In specific embodiments, a modified nucleoside is5′-O-(l-Thiophosphate)-Adenosine, 5′-O-(1-Thiophosphate)-Cytidine,5′-O-(1-Thiophosphate)-Guanosine, 5′-O-(1-Thiophosphate)-Uridine or5′-O-(l-Thiophosphate)-Pseudouridine.

In further specific embodiments the modified nucleotides includenucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine,α-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine,deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine,α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine,8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine,2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cytidine,6-Chloro-purine, N6-methyl-adenosine, α-thio-adenosine,8-azido-adenosine, 7-deaza-adenosine.

Further modified nucleotides have been described previously (WO2013/052523).

Sample: As used herein, the term “sample” refers to a liquid compositioncomprising the RNA to be purified and one or more impurities. The samplemay be the in vitro transcription mixture or it may be a partiallypurified sample. For example, the sample may have already been subjectedto any known RNA purification technique, in particular to an RP-HPLCpurification step and/or precipitation steps.

Impurity/impurities: The term “impurity” includes any molecule presentin the sample containing RNA other than the RNA to be purified. Inparticular, it includes components of the RNA in vitro transcriptionreaction such as enzymes, proteins and nucleotides.

Equilibration buffer: The term “equilibration buffer” refers to a saltsolution which is used to prepare the support material for loading thesample containing RNA. Within the method of the present invention theequilibration buffer is also used to load the sample containing RNA onthe support material. Therefore, it is passed through the supportmaterial simultaneously or substantially simultaneously with passage ofthe sample through the support material. In certain embodiments, theequilibration buffer is combined with the sample containing RNA prior topassage through the support material.

Support material: In chromatographic processes the support material istypically a material which serves as the stationary phase, i.e. as amaterial along which the mobile phase containing the molecules to beseparated, in the present case the equilibration buffer with the samplecontaining RNA, moves. The support material can be functionalized withligands which are suitable for the kind of separation desired. Forexample, for ion exchange chromatography the support material may befunctionalized with positively or negatively charged ligands and forhydrophobic interaction chromatography the support material may befunctionalized with hydrophobic ligands such as alkyl groups, arylgroups or combinations thereof.

The most widely used support materials are hydrophilic carbohydratessuch as cross-linked agarose and synthetic copolymer materials andmethacrylate based connective interaction media (CIM) monolithiccolumns. The support material may also comprise derivatives ofcellulose, polystyrene, synthetic poly amino acids, syntheticpolyacrylamide gels, cross-linked dextran or a glass surface.

For hydrophobic interaction chromatography or purification under highsalt conditions a hydrophobic ligand such as NH₂, SO₃H, PO₄H₂, SH,imidazoles, phenolic groups, butyl, hexyl, phenyl, octyl, polypropyleneglycol or non-ionic radicals such as OH and CONH₂ may be attached to thesupport material. These hydrophobic ligands may be attached usingdifunctional linking groups such as —NH—, —S— and —COO. Within thepresent invention, the use of OH and SO₃ ligands is particularlypreferred.

In some embodiments, the support material may be selected from the groupconsisting of an agarose media or a membrane functionalized with phenylgroups (e.g., Phenyl Sepharose™ from GE Healthcare or a Phenyl Membranefrom Sartorius), Tosoh Hexyl, CaptoPhenyl, Phenyl Sepharose™ 6 Fast Flowwith low or high substitution, Phenyl Sepharose™ High Performance, OctylSepharose™ High Performance (GE Healthcare); Fractogel™ EMD Propyl orFractogel™ EMD Phenyl (E. Merck, Germany); Macro-Prep™ Methyl orMacro-Prep™ t-Butyl columns (Bio-Rad, California); WP HI-Propyl (C3)™(J. T. Baker, New Jersey) or Toyopearl™ ether, phenyl or butyl(TosoHaas, PA). ToyoScreen PPG, ToyoScreen Phenyl, ToyoScreen Butyl, andToyoScreen Hexyl are based on rigid methacrylic polymer beads. GEHiScreen Butyl FF and HiScreen Octyl FF are based on high flow agarosebased beads. Preferred are Toyopearl Ether-650M, Toyopearl Phenyl-650M,Toyopearl Butyl-650M, Toyopearl Hexyl-650C (TosoHaas, PA), POROS-OH(ThermoFisher) or methacrylate based monolithic columns such as CIM-OH,CIM-SO₃, CIM-C4 A and CIM C4 HDL which comprise OH, sulfate or butylligands, respectively (BIA Separations).

The support material is preferably present in a column, wherein thesample containing RNA is loaded on the top of the column and the eluentis collected at the bottom of the column.

Washing buffer: The term “washing buffer”, as used herein refers to abuffer which is passed through the support material after loading thesample containing RNA and before eluting the RNA. The washing buffertherefore serves to remove impurities from the support material beforethe RNA is eluted.

Elution solution: An elution solution is used to disrupt the interactionbetween the RNA and the support material. Accordingly, the elutionsolution has a lower salt concentration than the equilibration bufferand the washing buffer.

Anion exchange chromatography: In anion exchange chromatography bindingto a support material is achieved by electrostatic interaction ofnegatively charged sample components with positively charged moietiessuch as diethylaminoethyl (DEAE) or quaternary ammonium (QA) on thesurface of the support material. This interaction typically occurs atlow salt concentrations and elution is achieved by increasing the saltconcentration.

Polar interaction chromatography: Polar interaction chromatography orhydrophilic interaction chromatography (HILIC) is based on theinteraction of components of a sample with a support material whichcarries polar functional groups such as hydroxyl or amine. Theinteraction may occur at high salt concentrations in the presence of anorganic solvent, in particular acetonitrile.

Hydrophobic interaction chromatography: Hydrophobic interactionchromatography is based on the hydrophobic interaction betweenhydrophobic moieties bound to a support material and hydrophobic regionsof the molecule that binds to the matrix such as RNA. Binding isachieved at high salt concentrations and the molecule is eluted from thematrix by decreasing the salt concentration and, optionally, theaddition of organic solvents.

In vitro transcription: The terms “in vitro transcription” or “RNA invitro transcription” relate to a process wherein RNA is synthesized in acell-free system (in vitro). DNA, particularly plasmid DNA, a PCRamplified DNA or synthetic DNA, is used as template for the generationof RNA transcripts. RNA may be obtained by DNA-dependent in vitrotranscription of an appropriate DNA template, which according to thepresent invention is preferably a linearized plasmid DNA template. Thepromoter for controlling in vitro transcription can be any promoter forany DNA-dependent RNA polymerase. Particular examples of DNA-dependentRNA polymerases are the T7, T3, and SP6 RNA polymerases. A DNA templatefor in vitro RNA transcription may be obtained by cloning of a nucleicacid, in particular cDNA corresponding to the respective RNA to be invitro transcribed, and introducing it into an appropriate vector for invitro transcription, for example into plasmid DNA. In a preferredembodiment of the present invention the DNA template is linearized witha suitable restriction enzyme, before it is transcribed in vitro. ThecDNA may be obtained by reverse transcription of mRNA or chemicalsynthesis. Moreover, the DNA template for in vitro RNA synthesis mayalso be obtained by gene synthesis or PCR.

Methods for in vitro transcription are known in the art (Geall et al.(2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) MethodsEnzymol. 530:101-14). Reagents used in said method typically include:

1) a DNA template with a promoter sequence that has a high bindingaffinity for its respective RNA polymerase such as bacteriophage-encodedRNA polymerases;

2) ribonucleoside triphosphates (NTPs) for the four bases (adenine,cytosine, guanine and uracil);

3) optionally a cap analog as defined below (e.g. m7G(5′)ppp(5′)G(m7G));

4) a DNA-dependent RNA polymerase capable of binding to the promotersequence within the linearized DNA template (e.g. T7, T3 or SP6 RNApolymerase);

5) optionally a ribonuclease (RNase) inhibitor to inactivate anycontaminating RNase;

6) optionally a pyrophosphatase to degrade pyrophosphate, which mayinhibit transcription;

7) MgCl₂, which supplies Mg²⁺ ions as a co-factor for the polymerase;

8) a buffer to maintain a suitable pH value, which can also containantioxidants (e.g. DTT), amines such as betaine and/or polyamines suchas spermidine at optimal concentrations.

“In vitro transcribed RNA” is an RNA which has been prepared by theprocess of in vitro transcription as described above.

DNA template: The DNA template provides the nucleic acid sequence whichis transcribed into the RNA by the process of in vitro transcription andwhich therefore comprises a nucleic acid sequence which is complementaryto the RNA sequence which is transcribed therefrom. In addition to thenucleic acid sequence which is transcribed into the RNA the DNA templatecomprises a promoter to which the RNA polymerase used in the in vitrotranscription process binds with high affinity.

Preferably, the DNA template may be a linearized plasmid DNA template.The linear template DNA is obtained by contacting plasmid DNA with arestriction enzyme under suitable conditions so that the restrictionenzyme cuts the plasmid DNA at its recognition site(s) and disrupts thecircular plasmid structure. The plasmid DNA is preferably cutimmediately after the end of the sequence which is to be transcribedinto RNA. Hence, the linear template DNA comprises a free 5′ end and afree 3′ end which are not linked to each other. If the plasmid DNAcontains only one recognition site for the restriction enzyme, thelinear template DNA has the same number of nucleotides as the plasmidDNA. If the plasmid DNA contains more than one recognition site for therestriction enzyme, the linear template DNA has a smaller number ofnucleotides than the plasmid DNA. The linear template DNA is then thefragment of the plasmid DNA which contains the elements necessary for invitro transcription, that is a promotor element for RNA transcriptionand the template DNA element. The open reading frame of the lineartemplate DNA determines the sequence of the transcribed RNA by the rulesof base-pairing.

In other embodiments, the DNA template may be selected from a syntheticdouble stranded DNA construct, a single-stranded DNA template with adouble-stranded DNA region comprising the promoter to which the RNApolymerase binds, a cyclic double-stranded DNA template with promoterand terminator sequences or a linear DNA template amplified by PCR orisothermal amplification.

Monolithic support material: A monolithic support material (ormonolithic bed) is a continuous bed consisting of a single piece of ahighly porous solid material where the pores are highly interconnectedforming a network of flow-through channels. Hence, the void volume isdecreased to a minimum and all the mobile phase is forced to flowthrough the large pores of the medium.

Three types of monolithic support materials are commercially available:

1) Silica gel based monolithic beds which are solid rods of silicamonolith that have been prepared according to a sol-gel process. Thisprocess is based on the hydrolysis and polycondensation of alkoxysilanesin the presence of water-soluble polymers. The method leads to “rods”made of a single piece of porous silica with a defined bimodal porestructure having macro (of about 2 μm) and mesopores (of about 0.013 μm)when smaller rods intended for analytical purposes are prepared.

2) Polyacrylamide based monolithic beds are made of swollenpolyacrylamide gel compressed in the shape of columns. Their technologyrelies on the polymerization of advanced monomers and ionomers directlyin the chromatographic column. In the presence of salt, the polymerchains form aggregates into large bundles by hydrophobic interaction,creating voids between the bundles (irregularly shaped channels) largeenough to permit a high hydrodynamic flow.

3) Rigid organic gel based monolithic beds: These supports are preparedby free radical polymerization of a mixture of a polymerizable monomer,optionally with functional groups, such as glycidyl methacrylate,ethylene dimethacrylate, a crosslinking agent, a radical chaininitiator, such as 2,2′-azobisisobutyronitrile, and porogenic solvents(cyclohexanol and dodecanol) in barrels of an appropriate mold (Svec F,Tennikova T B (1991) J Bioact Compat Polym 6: 393; Svec F, Jelinkova M,Votavova E (1991) Angew Macromol Chem 188: 167; Svec F, Frechet J M J(1992) Anal Chem 64: 820) in the case of glycidylmethacrylate-co-ethylene dimethacrylate (GMA-EDMA) monoliths.

DNase: DNases are enzymes which hydrolyze DNA by that catalyzing thehydrolytic cleavage of phosphodiester linkages in the DNA backbone.Suitable DNases are isolated from bovine pancreas and are available fromvarious suppliers such as Sigma-Aldrich, New England Biolabs, Qiagen andThermoFisher. Preferably, the used DNase is free of any RNAse activity.In one embodiment the treatment with DNase is performed in DNase bufferadditionally comprising a suitable amount of calcium chloride, such as0.66 mM CaCl₂. The DNA is treated with the DNase for 1 to 5 hours,preferably for 1.5 to 3 hours and more preferably for 2 hours. The DNasetreatment is preferably performed at a temperature of 37° C. In oneparticular embodiment, the DNA template is removed by addition of 0.66mM CaCl₂ and 300 U/ml DNase I in digestion buffer and incubation for twohours at 37° C. The DNase treatment can be stopped by adding EDTA oranother chelating agent. Preferably, the DNase treatment is stopped byadding EDTA to a final concentration of 25 mM.

HPLC: HPLC is the common abbreviation of the term “high performanceliquid chromatography”. In the HPLC process a pressurized liquid solventcontaining the sample mixture is passed through a column filled with asolid adsorbent material leading to the interaction of components of thesample with the adsorbent material. Since different components interactdifferently with the adsorbent material, this leads to the separation ofthe components as they flow out of the column. The operational pressurein HPLC process is typically between 50 and 350 bar. The term HPLCincludes reversed phase HPLC (RP-HPLC), size exclusion chromatography,gel filtration, affinity chromatography, hydrophobic interactionchromatography or ion pair chromatography, wherein reversed phase HPLCis preferred.

Reversed phase HPLC (RP-HPLC): Reversed phase HPLC uses a non-polarstationary phase and a moderately polar mobile phase and therefore workswith hydrophobic interactions which result from repulsive forces betweena relatively polar solvent, the relatively non-polar analyte, and thenon-polar stationary phase (reversed phase principle). The retentiontime on the column is therefore longer for molecules which are morenon-polar in nature, allowing polar molecules to elute more readily. Theretention time is increased by the addition of polar solvent to themobile phase and decreased by the addition of more hydrophobic solvent.

The characteristics of the specific RNA molecule as an analyte may playan important role in its retention characteristics. In general, ananalyte having more apolar functional groups results in a longerretention time because it increases the molecule's hydrophobicity andtherefore the interaction with the non-polar stationary phase. Verylarge molecules, however, can result in incomplete interaction betweenthe large analyte surface and the alkyl chain. Retention time increaseswith hydrophobic surface area which is roughly inversely proportional tosolute size. Branched chain compounds elute more rapidly than theircorresponding isomers because the overall surface area is decreased.

Ion-pair, reversed-phase HPLC: Ion-pair, reversed-phase HPLC is aspecific form of reversed-phase HPLC in which an ion with a lipophilicresidue and positive charge such as an alkylammonium salt, e.g.triethylammonium acetate, is added to the mobile phase as counter ionfor the negatively charged RNA. When used with common hydrophobic HPLCphases in the reversed-phase mode, ion pair reagents can be used toselectively increase the retention of the RNA.

Pharmaceutical composition: A pharmaceutical composition is acomposition comprising a pharmaceutically active agent such as atherapeutic RNA and one or more pharmaceutically acceptable carriers. Apharmaceutical composition is suitable for storage for a certain periodof time and for administration to a patient. The pharmaceuticalcomposition may be in liquid or in freeze-dried form. A suitableinjection solution for RNA is disclosed in WO 2006/122828 A2. A methodfor lyophilizing RNA is described in WO 2016/165831 A1. Thepharmaceutical composition comprises the RNA in a pharmaceuticallyeffective amount which is able to exert the therapeutic effect. The RNAmay be complexed using cationic and/or polycationic compounds such aspolycationic peptides or polymers (see, e.g., WO 2009/030481 A1; WO2012/013326 A1; WO 2013/113501 A1; WO 2013/113736 A1) or may beencapsulated (e.g., lipid nanoparticles (LNPs), liposomes).

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, the present invention is based on the finding thatRNA can be purified either from a crude in vitro transcription mixtureor from an RP-HPLC-purified mixture by a chromatography step wherein theRNA binds to the support material or the moiety attached thereto underhigh salt conditions and is then eluted by decreasing the saltconcentration. These conditions are also used in hydrophobic interactionchromatography so that the method of the present invention may involvehydrophobic interaction chromatography, although the support materialused in the method of the present invention is not restricted to thematerial typically used in hydrophobic interaction chromatography, butmay also comprise material which is typically used in otherchromatographic techniques such as ion exchange chromatography. Oneexample of such a material is a support material with sulfate groups. Inthe present invention the binding of the RNA to the support materialdoes not involve the interaction between nucleotide bases within the RNAand nucleotide bases attached to the support, in particular the bindingof the RNA to the support material does not involve the interactionbetween the polyA tail of the RNA and thymidines attached to thesupport.

Hence, in a first aspect, the present invention relates to a method forpurifying RNA, comprising the steps of:

a) applying a sample containing RNA in an equilibration buffer having ahigh salt concentration to a support material capable of binding the RNAunder high salt conditions, wherein the support comprises hydroxyl orsulfate groups;

b) washing the support material with a washing buffer having a high saltconcentration; and

c) eluting the nucleic acid from the support material with an elutionsolution,

wherein the method does not comprise a polar interaction chromatographyor an anion exchange chromatography step.

In another aspect the present invention relates to a method forpurifying in vitro transcribed RNA, comprising the steps of:

a) transcribing RNA from a template DNA in vitro;

b) applying a sample containing the in vitro transcribed RNA in anequilibration buffer having a high salt concentration to a supportmaterial capable of binding the RNA under high salt conditions, whereinthe support comprises hydroxyl or sulfate groups;

c) washing the support material with a washing buffer having a high saltconcentration; and

d) eluting the RNA from the support material with an elution solution.

Before applying the sample containing RNA or in vitro transcribed RNA tothe support material, the sample may be diluted, for example withequilibration buffer. Preferably, the sample is diluted between 1:2 and1:20, more preferably it is diluted between 1:5 and 1:12 and mostpreferably it is diluted 1:10, i.e. one volume of the sample is mixedwith 9 volumes of equilibration buffer. In other embodiments, the samplecontaining RNA or in vitro transcribed RNA is not diluted withequilibration buffer before applying the sample to the support material.

In the process of the present invention, any monolithic support can beused which is permeable for RNA. Preferably, the monolithic support isbased on a methacrylate polymer, more preferably it is based onpoly(glycidyl methacrylate-co ethylene dimethylacrylate). The averagepore radius is preferably 500 to 1200 nm, preferably it is 675 nm. Alsopreferably the monolithic support is CIM® available from BIASeparations.

As described above, the monolithic bed may carry functional moieties(ligands) that allow for the specific chromatographic separation. Theligand density is chosen such that capacity, yield and recovery aremaximized.

Preferably, the monolithic bed comprises a hydroxyl or a sulfate moietyand more preferably it is CIM® OH or CIM® SO₃ available from BIASeparations. The hydroxyl moiety is attached to the monolithic beddirectly. In particular, the hydroxyl moiety is not part of a ligandcarrying additional chemical groups such as the ligand N-benzylethanolamine.

The solution applied to the support material has an RNA concentration of0.05 mg/ml to 5 mg/ml, preferably of 0.07 mg/ml to 3 mg/ml, morepreferably of 0.1 mg/ml to 1 mg/ml or 0.1 mg/ml to 0.5 mg/ml and mostpreferably the RNA concentration is 0.2 mg/ml.

The equilibration buffer has a high salt concentration to enhance theinteraction of the RNA with the support material or the ligand attachedthereto. Preferably, the high salt concentration is from 50 mM to 5 M orfrom 100 mM to 4 M, more preferably, the salt concentration is from 300mM to 3.5 M or from 500 mM to 3 M, even more preferably the high saltconcentration is from 700 mM to 2.8 M or from 1.2 M to 2.5 M and mostpreferably it is 2 M, depending, in part, on the salt type. In oneembodiment the high salt concentration is 1 M, 1.1 M, 1.2 M, 1.3 M, 1.4M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M,2.5 M, 2.6 M, 2.7 M, 2.8 M, 2.9 M or 3.0 M.

The equilibration buffer may comprise a salt selected from the groupconsisting of sodium chloride, ammonium sulfate, sodium sulfate,ammonium chloride, sodium bromide, sodium citrate or a combinationthereof. In a particular embodiment, the equilibration buffer comprisessodium chloride. The equilibration buffer may comprise a cation selectedfrom the group consisting of Ba²⁺, Ca²⁺, Mg²⁺, Li⁺, Cs⁺, Na⁺, K⁺, Rb⁺,and NH₄ ⁺, and/or an anion selected from the group consisting of PO₄ ³⁻,SO₄ ²⁻, CH₃CO₃ ⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻, I⁻, and SCN⁻ or a combinationthereof.

In a preferred embodiment the equilibration buffer comprises 2 M sodiumchloride.

The pH of the equilibration buffer is between 4.0 and 8.5 or between 5.0and 8.0. In certain embodiments, the equilibration buffer has a pHbetween 6.0 and 7.5. Most preferably, the pH of the equilibration bufferis 7.0.

The equilibration buffer may contain a buffer substance which is a weakacid or base used to maintain the acidity (pH) of a solution near achosen value after the addition of another acid or base. Hence, thefunction of a buffer substance is to prevent a rapid change in pH whenacids or bases are added to the solution. Suitable buffer substances foruse in the present invention are HEPES(2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), Tris(2-amino-2-hydroxymethyl-propane-1,3-diol), phosphate buffer and acetatebuffer.

Most preferably, the equilibration buffer comprises 20 mM HEPES-NaOH, pH7.0 and 2 M NaCl.

Preferably, the equilibration buffer does not contain 1 mM EDTA and morepreferably it does not contain any EDTA at all.

The washing buffer has a high salt concentration so that the interactionof the RNA with the support material or the ligand attached thereto isnot interrupted during washing. Preferably, the high salt concentrationis from 50 mM to 5 M or from 100 mM to 4 M, more preferably, the saltconcentration is from 300 mM to 3.5 M or from 500 mM to 3 M, even morepreferably the high salt concentration is from 700 mM to 2.8 M or from1.2 M to 2.5 M and most preferably it is 2 M, depending, in part, on thesalt type. In one embodiment the high salt concentration is 1 M, 1.1 M,1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M,2.2 M, 2.3 M, 2.4 M, 2.5 M, 2.6 M, 2.7 M, 2.8 M, 2.9 M or 3.0 M.

The washing buffer may comprise a salt selected from the groupconsisting of sodium chloride, ammonium sulfate, sodium sulfate,ammonium chloride, sodium bromide or a combination thereof. In aparticular embodiment, the equilibration buffer comprises sodiumchloride. The washing buffer may comprise a cation selected from thegroup consisting of Ba²⁺, Ca²⁺, Mg²⁺, Li⁺, Cs⁺, Na⁺, K⁺, Rb⁺, and NH₄ ⁺,and/or an anion selected from the group consisting of PO₄ ³⁻, SO₄ ²⁻,CH₃CO₃ ⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻, I⁻, and SCN⁻ or a combinationthereof.

In a preferred embodiment the washing buffer comprises 2 M sodiumchloride.

The pH of the washing buffer is between 4.0 and 8.5 or between 5.0 and8.0. In certain embodiments, the equilibration buffer has a pH between6.0 and 7.5. Most preferably, the pH of the washing buffer is 7.0.

The washing buffer may contain a buffer substance which is a weak acidor base used to maintain the acidity (pH) of a solution near a chosenvalue after the addition of another acid or base. Hence, the function ofa buffer substance is to prevent a rapid change in pH when acids orbases are added to the solution. Suitable buffer substances for use inthe present invention are HEPES(2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), Tris(2-amino-2-hydroxymethyl-propane-1,3-diol), phosphate buffer and acetatebuffer.

More preferably, the washing buffer has the same composition and pH asthe equilibration buffer. Most preferably, the washing buffer comprises20 mM HEPES-NaOH, pH 7.0 and 2 M NaCl.

Preferably, the washing buffer does not contain 1 mM EDTA and morepreferably it does not contain any EDTA at all.

The RNA is eluted from the support material by a gradually decreasingsalt gradient. To this end, the percentage of the elution solution whichis in contact with the support material is gradually increased, therebydisrupting the interaction between the RNA and the support material.

The flow rate of the elution solution is selected such that goodseparation of the RNA from the impurities contained in the sample isachieved. The eluent flow rate may amount to from 0.5 ml/min to 5ml/min, preferably from 1 ml/min to 4 ml/min, more preferably it is 3ml/min. This flow rate may be established and regulated by a pump.

The eluent flow rate is also dependent on the volume of the used column(CV). The flow rate may amount to from 1.5 CV/min to 15 CV/min,preferably from 3 CV/min to 12 CV/min, more preferably it is 9 CV/min.This flow rate may be established and regulated by a pump.

The elution solution may have a salt concentration of less than 500 mM,if the equilibration buffer and the washing buffer have a saltconcentration of at least 1 M. The elution solution may have a saltconcentration of less than 200 mM, if the equilibration buffer and thewashing buffer have a salt concentration of at least 500 mM. The elutionsolution may have a salt concentration of less than 100 mM, if theequilibration buffer and the washing buffer have a salt concentration ofat least 300 mM. The elution solution may have a salt concentration ofless than 50 mM, if the equilibration buffer and the washing buffer havea salt concentration of at least 150 mM. The elution solution may have asalt concentration of less than 20 mM, if the equilibration buffer andthe washing buffer have a salt concentration of at least 100 mM.

In one embodiment, the elution solution does not comprise any salt.

In one embodiment, the elution solution is water. In another embodiment,the elution solution comprises a buffer substance selected from thegroup consisting of HEPES(2-[4-(2-hydroxyethyl)-piperazin-1-yl]ethanesulfonic acid), Tris(2-amino-2-hydroxymethyl-propane-1,3-diol), citrate buffer, phosphatebuffer and acetate buffer.

The pH of the elution solution is between 4.0 and 8.5 or between 5.0 and8.0. In certain embodiments, the elution solution has a pH between 6.0and 7.5. Most preferably, the pH of the washing buffer is 7.0.

In one embodiment, the elution solution comprises the same buffersubstance as the equilibration buffer and/or the washing buffer, but hasa lower salt concentration as the equilibration buffer and/or thewashing buffer as described above. In one embodiment, the elutionsolution comprises the same buffer substance as the equilibration bufferand/or the washing buffer, but does not comprise any salt. In oneembodiment, the elution solution has the same pH as the equilibrationbuffer and/or the washing buffer, but has a lower salt concentration asthe equilibration buffer and/or the washing buffer as described above.In one embodiment, the elution solution has the same pH as theequilibration buffer and/or the washing buffer, but does not compriseany salt. In one embodiment, the elution solution comprises the samebuffer substance as the equilibration buffer and/or the washing bufferand has the same pH as the equilibration buffer and/or the washingbuffer, but has a lower salt concentration as the equilibration bufferand/or the washing buffer as described above. In one embodiment, theelution solution comprises the same buffer substance as theequilibration buffer and/or the washing buffer and has the same pH asthe equilibration buffer and/or the washing buffer, but does notcomprise any salt. In a preferred embodiment the elution solutioncomprises 20 mM HEPES-NaOH, pH 7.0.

Preferably, the elution solution does not contain 1 mM EDTA and morepreferably it does not contain any EDTA at all.

The RNA which is eluted from the support material is preferably detectedby UV measurement at 260 nm.

In one embodiment, the method of the present invention comprises anadditional purification step, before the RNA is subjected to thechromatography under high salt conditions as claimed herein. Theadditional purification step is preferably a RP-HPLC step. Aparticularly preferred method for purifying the target RNA by RP-HPLC isdisclosed in WO 2008/077592 A1 and involves a reversed-phase HPLC usinga porous reversed phase as stationary phase.

In one embodiment, the HPLC fraction comprising RNA obtained fromRP-HPLC is subjected to the chromatography under high salt conditions asclaimed herein.

In another embodiment, the HPLC fraction comprising RNA is subjected toa precipitation step to remove acetonitrile and triethylammonium acetatebefore it is subjected to the chromatography under high salt conditionsas claimed herein.

In general, any material known to be used as reverse phase stationaryphase, in particular any polymeric material may be used, if thatmaterial can be provided in porous form. The stationary phase may becomposed of organic and/or inorganic material. Examples for polymers tobe used for the purification step of the present invention are(non-alkylated) polystyrenes, (non-alkylated)polystyrenedivinylbenzenes, silica gel, silica gel modified withnon-polar residues, particularly silica gel modified with alkylcontaining residues, more preferably with butyl-, octyl and/or octadecylcontaining residues, silica gel modified with phenylic residues,polymethacrylates, etc.

In a particularly preferred embodiment, the material for the reversedphase is a porous polystyrene polymer, a (non-alkylated) porouspolystyrenedivinylbenzene polymer, porous silica gel, porous silica gelmodified with non-polar residues, particularly porous silica gelmodified with alkyl containing residues, more preferably with butyl-,octyl and/or octadecyl containing residues, porous silica gel modifiedwith phenylic residues, porous polymethacrylates, wherein in particulara porous polystyrene polymer or a non-alkylated (porous)polystyrenedivinylbenzene may be used.

A non-alkylated porous polystyrenedivinylbenzene which is particularlypreferred for the RP-HPLC step is one which, without being limitedthereto, may have a particle size of 8.0±1.5 μm, in particular 8.0±0.5μm, and a pore size of 1000-1500 Å, in particular 1000-1200 Å or3500-4500 Å.

The stationary phase is conventionally located in a column. V2A steel isconventionally used as the material for the column, but other materialsmay also be used for the column provided they are suitable for theconditions prevailing during HPLC. Conventionally the column isstraight. It is favourable for the HPLC column to have a length of 5 cmto 100 cm and a diameter of 4 mm to 25 mm. Columns used for thepurification step of the method of the invention may in particular havethe following dimensions: 50 mm long and 7.5 mm in diameter or 50 mmlong and 4.6 mm in diameter, or 50 mm long and 10 mm in diameter or anyother dimension with regard to length and diameter, which is suitablefor preparative recovery of RNA, even lengths of several meters and alsolarger diameters being feasible in the case of upscaling.

The HPLC is preferably performed as ion-pair, reversed phase HPLC asdefined above.

In a preferred embodiment, a mixture of an aqueous solvent and anorganic solvent is used as the mobile phase for eluting the RNA.Preferably, the buffer used as the aqueous solvent has a pH of 6.0-8.0,for example of about 7, for example 7.0. More preferably the buffer istriethylammonium acetate which preferably has a concentration of 0.02 Mto 0.5 M, more preferably of 0.08 M to 0.12 M. Most preferably, an 0.1 Mtriethylammonium acetate buffer is used, which also acts as a counterion to the RNA in the ion pair method.

In a preferred embodiment, the organic solvent which is used in themobile phase is selected from acetonitrile, methanol, ethanol,1-propanol, 2-propanol and acetone or a mixture thereof. More preferablyit is acetonitrile.

In a particularly preferred embodiment, the mobile phase is a mixture of0.1 M triethylammonium acetate, pH 7, and acetonitrile.

Preferably, the mobile phase contains 5.0 vol. % to 25.0 vol. % organicsolvent, relative to the mobile phase, and for this to be made up to 100vol. % with the aqueous solvent. Typically, in the event of gradientseparation, the proportion of organic solvent is increased, inparticular by at least 10%, more preferably by at least 50% and mostpreferably by at least 100%, optionally by at least 200%, relative tothe initial vol. % in the mobile phase. In a preferred embodiment, theproportion of organic solvent in the mobile phase amounts in the courseof HPLC separation to 3 to 9, preferably 4 to 7.5, in particular 5.0vol. %, in each case relative to the mobile phase. More preferably, theproportion of organic solvent in the mobile phase is increased in thecourse of HPLC separation from 3 to 9, in particular 5.0 vol. % to up to20.0 vol. %, in each case relative to the mobile phase. Still morepreferably, the method is performed in such a way that the proportion oforganic solvent in the mobile phase is increased in the course of HPLCseparation from 6.5 to 8.5, in particular 7.5 vol. %, to up to 17.5 vol.%, in each case relative to the mobile phase.

Even more preferably the mobile phase contains 7.5 vol. % to 17.5 vol. %organic solvent, relative to the mobile phase, and for this to be madeup to 100 vol. % with the aqueous buffered solvent.

Elution may proceed isocratically or by means of gradient separation. Inisocratic separation, elution of the RNA proceeds with a single eluentor a constant mixture of a plurality of eluents, wherein the solventsdescribed above in detail may be used as eluent.

In a preferred embodiment, gradient separation is performed wherein thecomposition of the eluent is varied by means of a gradient program. Theequipment necessary for gradient separation is known to a person skilledin the art. Gradient elution may here proceed either on the low pressureside by mixing chambers or on the high pressure side by further pumps.

Preferably, the proportion of organic solvent, as described above, isincreased relative to the aqueous solvent during gradient separation.The above-described agents may here be used as the aqueous solvent andthe likewise above-described agents may be used as the organic solvent.For example, the proportion of organic solvent in the mobile phase maybe increased in the course of HPLC separation from 5.0 vol. % to 20.0vol. %, in each case relative to the mobile phase. In particular, theproportion of organic solvent in the mobile phase may be increased inthe course of HPLC separation from 7.5 vol. % to 17.5 vol. %, inparticular 9.5 to 14.5 vol. %, in each case relative to the mobilephase.

The following gradient program has proven particularly favourable forthe purification of RNA:

Eluent A: 0.1 M triethylammonium acetate, pH 7

Eluent B: 0.1 M triethylammonium acetate, pH 7, with 25 vol. %acetonitrile

Eluent Composition:

-   -   start: 62% A and 38% B (1st to 3rd minute)    -   increase to 58% B (1.67% increase in B per minute), (3rd-15th        minute)    -   100% B (15th to 20th minute)

Another example of a gradient program is described below, the sameeluent A and B being used:

Eluent Composition:

-   -   starting level: 62% A and 38% B (1st-3rd min)    -   separation range I: gradient 38%-49.5% B (5.75% increase in        B/min) (3rd-5th min)    -   separation range II: gradient 49.5%-57% B (0.83% increase in        B/min) (5th-14th min)    -   rinsing range: 100% B (15th-20th min)

It is preferred to use purified solvent for HPLC. Such purified solventsare commercially obtainable. They may additionally also be filteredthrough a 1 to 5 μm microfilter, which is generally mounted in thesystem upstream of the pump. It is additionally preferred for all thesolvents to be degassed prior to use, since otherwise gas bubbles occurin most pumps. If air bubbles occur in the solvent, they may interferenot only with separation but also with the continuous monitoring ofoutflow in the detector. The solvents may be degassed by heating, byvigorous stirring with a magnetic stirrer, by brief evacuation, byultrasonication or by passing a small stream of helium through thesolvent storage vessel.

The flow rate of the eluent is selected such that good separation of theRNA from the other constituents contained in the sample to beinvestigated takes place. The eluent flow rate may amount to from 1ml/min to several liters per minute (in the case of upscaling), inparticular about 1 to 1000 ml/min, more preferably 5 ml to 500 ml/min,even more preferably more than 100 ml/min, depending on the type andscope of the upscaling. This flow rate may be established and regulatedby the pump.

The HPLC is preferably performed under denaturing conditions, such as anincreased temperature. Suitable temperature conditions include atemperature of at least 70° C., preferably of at least 75° C., morepreferably of about 78° C. By using denaturing conditions anyintramolecular double strands formed between two RNA strands or betweenan RNA strand and a DNA strand are disrupted so that onlysingle-stranded nucleic acid molecules are present in the sample.

Detection proceeds preferably with a UV detector at 254 nm, wherein areference measurement may be made at 600 nm. However, any otherdetection method may alternatively be used, with which the RNA may bedetected.

For preparative purification of the RNA, it is advisable to collect theRNA-containing eluted solvent quantities. In this respect, it ispreferred to carry out this collection in such a way that the elutedsolvent is collected in individual separated fractions. This may takeplace for example with a fraction collector. In this way, thehigh-purity RNA-containing fractions may be separated from otherRNA-containing fractions which still contain undesired impurities,albeit in very small quantities. The individual fractions may becollected for example over 1 minute.

The HPLC is preferably performed under completely denaturing conditions.This may proceed for example in that sample application takes place at atemperature of 4-12° C., the HPLC method otherwise proceeding at ahigher temperature, preferably at 70° C. or more, particularlypreferably at 75° C. or more, in particular up to 82° C., and veryparticularly preferably at about 78° C.

Sample application may be performed with two methods, stop-flowinjection or loop injection. For stop-flow injection a microsyringe isused which is able to withstand the high pressure applied in HPLC. Thesample is injected through a septum in an inlet valve either directlyonto the column packing or onto a small drop of inert materialimmediately over the packing. The system may in this case be underelevated pressure, or the pump may be turned off prior to injection,which is then performed when the pressure has fallen to close to thenormal value. In the case of loop injection, a loop injector is used tointroduce the sample. This consists of a tubular loop, into which thesample is inserted. By means of a suitable rotary valve, the stationaryphase is then conveyed out of the pump through the loop, whose outletleads directly into the column. The sample is entrained in this way bythe stationary phase into the column, without solvent flow to the pumpbeing interrupted.

In a particularly preferred embodiment, the material for the reversedphase is a poly-styrenedivinylbenzene, wherein in particularnon-alkylated polystyrenedivinyl-benzene may be used. A non-alkylatedporous polystyrenedivinylbenzene which is very particularly is one whichhas in particular a particle size of 8.0±1.5 μm, in particular 8.0±0.5μm, and a pore size of 1000- or 4000 Å. With this material for thereversed phase, the advantages described below may be achieved in aparticularly favourable manner.

The eluate of the RP-HPLC step contains the RNA. The RNA in the eluateis purified as compared to the RNA sample subjected to the RP-HPLC step.

After the RP-HPLC step any organic solvent present in the eluate may beremoved by suitable methods which are known to the skilled person. Thesemethods include, but are not limited to, precipitation with isopropanolor lithium chloride, tangential flow filtration and dialysis. In apreferred embodiment the organic solvent is removed by precipitation ofthe RNA with isopropanol.

The purified RNA which is obtained by the method of the presentinvention can be used to prepare a pharmaceutical composition. Thepharmaceutical composition can be prepared by admixing the RNA with oneor more pharmaceutically acceptable carriers. Sterile injectable formsof the pharmaceutical composition may be aqueous or oleaginoussuspension. These suspensions may be formulated according to techniquesknown in the art using suitable dispersing or wetting agents andsuspending agents. A pharmaceutically acceptable carrier typicallyincludes the liquid or non-liquid basis of a composition comprising thecomponents of the composition. If the composition is provided in liquidform, the carrier will typically be pyrogen-free water; isotonic salineor buffered (aqueous) solutions, e.g. phosphate, citrate etc. bufferedsolutions. The injection buffer may be hypertonic, isotonic or hypotonicwith reference to the specific reference medium, i.e. the buffer mayhave a higher, identical or lower salt content with reference to thespecific reference medium, wherein preferably such concentrations of theafore mentioned salts may be used, which do not lead to damage of cellsdue to osmosis or other concentration effects. Reference media are e.g.liquids occurring in “in vivo” methods, such as blood, lymph, cytosolicliquids, or other body liquids, or e.g. liquids, which may be used asreference media in “in vitro” methods, such as common buffers orliquids. Such common buffers or liquids are known to a skilled person.Ringer-Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds, which are suitable for administration to apatient to be treated, may be used as well for the pharmaceuticalcomposition. The term “compatible” as used here means that theseconstituents of the inventive pharmaceutical composition are capable ofbeing mixed with the components of the pharmaceutical composition insuch a manner that no interaction occurs which would substantiallyreduce the pharmaceutical effectiveness of the pharmaceuticalcomposition under typical use conditions.

Although the method of the present invention is particularly suitablefor use in the context of small scale RNA purification, it may also beused with larger amounts of RNA such as several 100 grams of RNA.Preferably, the method of the present invention yields an amount ofpurified RNA of 0.1 g to 5 g, more preferably of 0.3 g to 3 g and mostpreferably of 0.5 g to 2 g. To obtain this amount of purified RNA 0.23 gto 11.6 g, preferably 0.69 g to 6.9 g and more preferably 1.16 g to 4.65g of RNA have to be subjected to the method of the present invention.

The method with the method steps as defined herein may not only be usedto purify RNA, but also to polish RNA preparations, i.e. to removeresidual impurities from a partially purified RNA sample, to concentratethe RNA preparation and to re-buffer the RNA preparation and to captureRNA present in a solution.

The present invention was made with support from the Government underAgreement No. HR0011-11-3-0001 awarded by DARPA. The Government hascertain rights in the invention.

EXAMPLES

The following Examples are merely illustrative and shall describe thepresent invention in a further way. The Examples shall not be construedto limit the present invention thereto.

Example 1 Preparation of RNA Solutions

1. Preparation of DNA and mRNA Constructs:

For the present Examples, a DNA sequence was prepared by modifying theDNA sequence by GC-optimization for stabilization. The GC-optimized DNAsequence was introduced into a pUC19 derived vector.

2. RNA In Vitro Transcription:

The obtained plasmid DNA was used for RNA in vitro transcriptionexperiments to obtain the RNA according to SEQ ID NO: 1.

The EcoRI linearized DNA plasmid was transcribed in vitro using T7polymerase. RNA in vitro transcription was performed in the presence ofa CAP analog (m7GpppG). RNA in vitro transcription was carried out in5.8 mM m7G(5′)ppp(5′)G Cap analog, 4 mM ATP, 4 mM CTP, 4 mM UTP, and1.45 mM GTP, 50 μg/ml DNA plasmid, 80 mM HEPES, 24 mM MgCl₂, 2 mMSpermidine, 40 mM DTT, 100 U/μg DNA T7 RNA polymerase, 5 U/μg DNApyrophosphatase, and 0.2 U/μl RNAse inhibitor. The in vitrotranscription reaction was incubated for 4.5 hours at 37° C.

To remove DNA template, 0.66 mM CaCl₂ and 300U/ml DNase1 (Thermo Fisher)was added and incubated in digestion buffer for 2 h at 37° C. Thedigestion reaction was stopped by adding EDTA to a final concentrationof 25 mM. In the following examples the obtained preparation is referredto as “crude RNA IVT reaction”.

Optionally, the crude RNA IVT reaction was HPLC purified usingPureMessenger® (CureVac, Tübingen, Germany; according to WO 2008/077592A1). HPLC-purified RNA eluates were precipitated using isopropanolprecipitation in order to remove organic solvent. The samples were mixedwith 5M NaCl and 100% isopropanol. After incubation at 4° C., thereaction vials were centrifuged, and supernatants were discarded. TheRNA pellets were washed with ethanol, centrifuged, and supernatant wasremoved. The obtained RNA pellets were dried for 30 minutes at roomtemperature and eventually re-suspended in 2 ml WFI.

In the following examples the purified RNA preparation is referred to as“HPLC purified RNA”.

Example 2 Purification of HPLC Purified RNA Using HydrophobicInteraction Chromatography (HIC)

1. Buffers and Basic Procedure:

1 ml HPLC purified RNA probe was mixed with 10 ml high salt bindingbuffer to obtain a diluted RNA solution (about 0.2 mg/ml). The CIM-OHcolumn was attached to the FPLC device (ÄKTA avant) and equilibratedwith 20 ml 50% high salt binding buffer. The maximal pressure was set to5 MPa. The flow rate was 3 ml/min. After loading of 2.5 ml probe ontothe CIM-OH column, the salt concentration was gradually reduced byadding low salt elution buffer. During the procedure, differentfractions were taken. Moreover, the flow through was collected. Both,the collected fractions and flow through were analyzed (SDS page,Agarose gel electrophoresis).

2. HIC Using a CIM-OH Column (2 M NaCl in High Salt Binding Buffer):

HPLC purified RNA (R2025) was used as probe. To purify/concentrateHPLC-purified RNA, a CIM-OH column (CIM-OH, 340 μl CV, BIA separations)was attached to the FPLC device (ÄKTA avant, GE Healthcare LifeSciences) purged with ddH20 and equilibrated (equilibration buffer: 20mM HEPES-NaOH, pH 7.0; 2M NaCl). Then, 2 mg/ml RNA (R2025) was diluted1:10 with equilibration buffer and 500 μg RNA was loaded onto therespective column with 2 ml min−1 and a maximum pressure of 5 MPa. Thecaptured RNA was eluted using a gradually decreasing salt gradient witha flow rate of 3 ml min−1 (elution buffer: 20 mM HEPES-NaOH, pH 7.0).The elution profile of the RNA is shown in FIG. 1. Shortly aftersubjecting the RNA sample to the CIM-OH column (1), unbound sample waseluted by washing with equilibration buffer (2) that potentiallycomprised contaminants (e.g. spermidine, proteins). While decreasing thesalt concentration via increasing the concentration of the low saltbuffer (elution buffer: 20 mM HEPES-NaOH, pH 7.0) (3) the RNA fractioneluted as a sharp and defined peak (4).

2. HIC Using CIM-SO3 Columns

To purify/concentrate HPLC-purified RNA, a CIM-SO₃ column (CIM-SO₃, 340μl CV, BIA separations) was attached to the FPLC device (ÄKTA avant, GEHealthcare Life Sciences) purged with ddH₂0 and equilibrated(equilibration buffer: 20 mM HEPES-NaOH, pH 7.0; 2M NaCl). Then, 2 mg/mlRNA (R2025) was diluted 1:10 with equilibration buffer and 500 μg RNAwas loaded onto the respective column with 2 ml min⁻¹ and a maximumpressure of 5 MPa. The captured RNA was eluted using a graduallydecreasing salt gradient with a flow rate of 3 ml min⁻¹ (elution buffer:20 mM HEPES-NaOH, pH 7.0). The elution profile of the RNA is shown inFIG. 2. Shortly after subjecting the RNA sample to the CIM-OH column(1), unbound sample was eluted by washing with equilibration buffer (2)that potentially comprised contaminants (e.g. spermidine, proteins).While decreasing the salt concentration via increasing the concentrationof the low salt buffer (elution buffer: 20 mM HEPES-NaOH, pH 7.0) (3)the RNA fraction eluted as a sharp and defined peak (4).

Result:

Unexpectedly, the results show that HIC is a suitable method forcapturing RNA from a HPLC purified RNA sample. Particularly suitable aremonolithic column materials (CIM) bearing —OH and SO₃ moieties as theyshow high binding capacity for large RNA molecules. The results suggestthat the inventive method may be broadly applicable for the purificationand also for the re-buffering, conditioning, cleaning, polishing,concentrating and/or capturing of various kinds of RNA preparations. Onefurther advantage of the used material (CIM monolith) is that saidmaterials have a large working pH range (pH 2-pH 13) allowing forcleaning-in place with e.g. alkaline cleaning solutions. Anotheradvantage of the used material (CIM monolith) is that those macroporousmonoliths also allow for large-scale preparations as these columns canbe used with high flow rates.

To evaluate if the inventive method also works for crude RNApreparations containing multiple contaminations, the inventive HICmethod was applied to purify crude RNA IVT samples (see Example 3).

Example 3 Purification of Non-Purified RNA from a Crude RNA IVT ReactionUsing Hydrophobic Interaction Chromatography (HIC)

To test if also crude IVT RNA samples (prepared according to Example 1)could be purified using the inventive HIC method, 200 μl of anon-purified IVT RNA sample (1.3 mg/ml) was diluted 1:10 inequilibration buffer and applied to a monolithic CIM column (CIM-OH; 2ml min⁻¹, maximum pressure of 5 MPa). Elution of the RNA was performedvia increasing the concentration of the low salt elution buffer (elutionbuffer: 20 mM HEPES-NaOH, pH 7.0). Detection was performed via UVmeasurement at 260 nm. The elution profile of the RNA is shown in FIG.3.

Shortly after subjecting the crude IVT RNA sample to the CIM-OH column(1), unbound flow through waste sample was eluted by washing withequilibration buffer (2) that comprised multiple protein contaminants(e.g. T7 RNA Polymerase, Pyrophosphatase, etc.) of the crude IVT RNAreaction. While decreasing the salt concentration via increasing theconcentration of the low salt buffer (elution buffer: 20 mM HEPES-NaOH,pH 7.0) (3) the RNA fraction eluted as a sharp and defined peak (4).During elution, samples from the flow through and 5 different fractionsafter applying the elution buffer were taken. Although the very highA260 nm signal of the flow-through peak is indicative for salts andother low molecular weight components of the IVT mix (e.g. DTT andnucleotides), SDS-PAGE with subsequent silver staining was performed inorder to detect proteins in single fractions (FIG. 4a ). Whereas noproteins could be detected in elution fractions, protein contaminationwas found in the flow-through fraction. Agarose gel electrophoresisanalysis of the fractions shows that RNA cannot be found in the flowthrough but accumulates after increase of elution buffer (FIG. 4b ).

Result:

The results show that the inventive HIC method is suitable forcapturing, purifying and re-buffering of an RNA sample containingmultiple contaminations (crude IVT RNA sample). The results indicatethat the method may be broadly applicable for the purification and alsofor the re-buffering, conditioning, cleaning, polishing, concentratingand/or capturing of RNA from various sources (e.g., crude RNApreparations, crude RP-HPLC reactions etc.).

FIGURE LEGENDS

FIG. 1:

Elution profiles of a HIC with a CIM-OH column of 0.5 μg HPLC purifiedRNA under decreasing salt concentrations. 1: RNA sample subjected toCIM-OH, 2: waste fraction, 3: gradual increase of elution buffer, 4: RNAfraction. A detailed description of the experiment is provided in theexample section, Example 2.

FIG. 2:

Elution profiles of a HIC with a CIM-SO3 column using 0.5 μg purifiedRNA under decreasing salt concentrations. 1: RNA sample subjected toCIM-SO3; 2: waste fraction; 3: gradual increase of elution buffer; 4:RNA fraction. A detailed description of the experiment is provided inthe example section, Example 2.

FIG. 3:

Elution profiles of a HIC with a CIM-OH column using 0.5 μg RNA underdecreasing salt concentrations. 1: RNA sample subjected to CIM-OH; 2:waste fraction; 3: gradual increase of elution buffer; 4: RNA fraction.Asterisks indicate fractions that were further analyzed (see FIG. 4). Adetailed description of the experiment is provided in the examplesection, Example 3.

FIG. 4:

(A) Analysis of flow-through (1) and five (2-6) elution fractions (asindicated in FIG. 3) via silver staining of SDS-PAGE (which stainsproteins and nucleic acids). (B) Agarose gel electrophoresis of the samesamples. A detailed description of the experiment is provided in theexample section, Example 3.

1. Method for purifying RNA, comprising the steps of: a) applying asample containing RNA in an equilibration buffer having a high saltconcentration to a support material capable of binding the RNA underhigh salt conditions, wherein the support comprises hydroxyl or sulfategroups; b) optionally washing the support material with a washing bufferhaving a high salt concentration; and c) eluting the nucleic acid fromthe support material with an elution solution.
 2. Method according toclaim 1, wherein the RNA is obtained by RNA in vitro transcription. 3.Method according to claim 1 or 2, wherein the equilibration bufferand/or the washing buffer has a salt concentration of 50 mM to 5M. 4.Method according to any one of the preceding claims, wherein theequilibration buffer and/or the washing buffer comprises sodium chlorideor ammonium sulfate.
 5. Method according to any one of the precedingclaims, wherein the equilibration buffer and/or the washing buffercomprises 2 M NaCl.
 6. Method according to any one of the precedingclaims, wherein the equilibration buffer and/or the washing buffercomprises 20 mM HEPES-NaOH, pH 7.0, 2 M NaCl.
 7. Method according to anyone of the preceding claims, wherein the equilibration buffer and thewashing buffer have the same composition and the same pH.
 8. Methodaccording to any one of the preceding claims, wherein the supportmaterial is a monolithic support material.
 9. Method according to anyone of the preceding claims, wherein the support material is amethacrylate polymer.
 10. Method according to any one of the precedingclaims, wherein the RNA is eluted by gradually decreasing the saltconcentration.
 11. Method according to any one of the preceding claims,wherein the elution solution does not contain a salt.
 12. Methodaccording to any one of the preceding claims, wherein the elutionsolution comprises 20 mM HEPES-NaOH, pH 7.0.
 13. Method for purifying invitro transcribed RNA, comprising the steps of: a) transcribing RNA froma template DNA in vitro; b) applying a sample containing the in vitrotranscribed RNA in an equilibration buffer having a high saltconcentration to a support material capable of binding the RNA underhigh salt conditions, wherein the support material comprises hydroxyl orsulfate groups; c) washing the support material with a washing bufferhaving a high salt concentration; and d) eluting the RNA from thesupport material with an elution solution.
 14. Method according to claim13, further comprising a step a1) of degrading the template DNA. 15.Method according to claim 14, wherein the template DNA is degraded bytreatment with DNase.
 16. Method according to any one of claims 13 to15, further comprising a step a2) of subjecting the in vitro transcribedRNA to an RP-HPLC step.
 17. Method according to any one of claims 13 to16, further comprising a step e) of preparing a pharmaceuticalcomposition comprising said RNA.
 18. Method according to any one ofclaims 13 to 17, wherein the equilibration buffer and/or the washingbuffer has a salt concentration of 50 mM to 5M.
 19. Method according toany one of claims 13 to 18, wherein the equilibration buffer and/or thewashing buffer comprises sodium chloride or ammonium sulfate.
 20. Methodaccording to any one of claims 13 to 19, wherein the equilibrationbuffer and/or the washing buffer comprises 2 M NaCl.
 21. Methodaccording to any one of claims 13 to 20, wherein the equilibrationbuffer and/or the washing buffer comprises 20 mM HEPES-NaOH, pH 7.0, 2 MNaCl.
 22. Method according to any one of claims 13 to 21, wherein theequilibration buffer and the washing buffer have the same compositionand the same pH.
 23. Method according to any one of claims 13 to 22,wherein the support material is a monolithic support material. 24.Method according to any one of claims 13 to 23, wherein the supportmaterial is a methacrylate polymer.
 25. Method according to any one ofclaims 13 to 24, wherein the RNA is eluted by gradually decreasing thesalt concentration.
 26. Method according to any one of claims 13 to 25,wherein the elution solution does not contain a salt.
 27. Methodaccording to any one of claims 13 to 26, wherein the elution buffercomprises 20 mM HEPES-NaOH, pH 7.0.
 28. Use of hydrophobic interactionchromatography for the purification of RNA obtained by RNA in vitrotranscription.